Mismatch repair is a major contributor to genetic stability. This pathway is of clinical interest because mutations in mismatch repair genes are the cause of 1 of the most prevalent forms of hereditary colon cancer, and have been implicated in the development of a subset (~15%) of sporadic tumors that can occur in a variety of tissues. Mismatch repair defects also have implications for cancer treatment because inactivation of the pathway renders cells resistant to the cytotoxic effects of certain anti-tumor drugs. The primary goals of this project are to elucidate the molecular nature of this DNA repair system as it functions in replication error correction and in the cellular response to DNA damage. Our aims are 4-fold: (1) 9 activities (MutSa, MutSB, MutLa, Exol, RFC, PCNA, RPA, HMGB1, and DNA polymerase 6) have been implicated in eukaryotic mismatch repair, but it is clear that additional, and as yet unknown, components play important regulatory roles in the reaction. We intend to isolate these activities and establish their functions in the reaction. (2) We have shown that subsets of the known human mismatch repair activities catalyze several partial reactions that account at least in part for the complexity of the overall reaction. We plan further characterization of the corresponding multi-protein"DNA assemblies with respect to molecular activities, stoichiometry, and conformation, as well as modulatory effects of adenine nucleotides on these parameters. (3) The structural basis of mismatch/DNA lesion recognition and processing by the human mismatch repair system will be addressed in collaborative X-ray crystallographic studies with Lorena Beese's laboratory. (4) MutSa and MutLa defects render mammalian cells resistant to the cytotoxic effects of SN1 DNA methylators, 6-thioguanine, and cisplatin. In an attempt to clarify the function of the human mismatch repair system in the DNA damage response, we will pursue in vitro experiments with the hope of elucidating the mechanism of MutSa-and MutLa-dependent activation of the ATR damage-signaling kinase in response to O6-methylguanine lesions. [unreadable] [unreadable] [unreadable]